The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6kDa) to an intermediate-sized product iSS (18.3kDa) and to the mature SS (16.3kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.
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September 2002
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Research Article|
September 15 2002
Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii
Cédric INVERNIZZI;
Cédric INVERNIZZI
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
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Jonathan IMHOF;
Jonathan IMHOF
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
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Gabriela BURKARD;
Gabriela BURKARD
1
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
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Katharina SCHMID;
Katharina SCHMID
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
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Arminio BOSCHETTI
Arminio BOSCHETTI
2
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
2To whom correspondence should be addressed, at Römerstrasse 1, CH-3047 Bremgarten, Switzerland (e-mail arminio.boschetti@bluewin.ch).
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Publisher: Portland Press Ltd
Received:
March 06 2002
Revision Received:
May 29 2002
Accepted:
June 05 2002
Accepted Manuscript online:
June 05 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (3): 989–998.
Article history
Received:
March 06 2002
Revision Received:
May 29 2002
Accepted:
June 05 2002
Accepted Manuscript online:
June 05 2002
Citation
Cédric INVERNIZZI, Jonathan IMHOF, Gabriela BURKARD, Katharina SCHMID, Arminio BOSCHETTI; Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii. Biochem J 15 September 2002; 366 (3): 989–998. doi: https://doi.org/10.1042/bj20020378
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